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Characterization and Analysis of Crym GENSAT BAC transgenic mice
GENSAT is NIH funded project that was initiated to generate BAC/EGFP transgenic
lines with the intention to provide genetic tools that would facilitate the study of the
central nervous system (CNS). We have take advantage of the availability of GENSAT
transgenic mice to address whether any of the transgenic lines that have been generated
would be appropriate to study renal development. The analysis here provides the kidney
research community with basic information as to the utility of GENSAT transgenic
strains in furthering the study of kidney development. As part of the GUDMAP
consortium, we have tested several strains from GENSAT at a single appropriate time
point (E15.5) and screened the mice for their ability to aid in the isolation of specific
components from the developing kidney for gene expression profiling. We have utilized
Crym-EGFP transgenic mice to examine the transcription profile of cells at E15.5 as well
as several other developmental time points including P0, P1, P2, P3 and P4. Here we
report the pattern of EGFP expression in the developing kidney of the Crym-EGFP strain.
Our analysis suggests that the Crym-EGFP transgenic mice may be a useful tool to
study the development of the cap mesenchyme and renal vesicle.
Crym Gene Notes
The crystallins are family of proteins that have been determined to have multiple roles.
One family member encodes the major proteins of vertebrate eye lens and maintains the
transparency and refractive index of the lens. Additional members do not perform
structural roles in lens tissue, but instead bind to thyroid hormone for possible regulatory
or developmental roles (Graw).
Strain Information
Strain Name: STOCK Tg(Crym-EGFP)82Gsat/Mmcd
Stock Number: 012003-UCD
Promoter: Crym
Name: crystallin, mu
Alteration at locus: Transgenic
Reporter: EGFP (Jelly Fish)
Name: Enhanced Green Fluorescent Protein
Alteration at locus: Transgenic
Genetic Alterations:
Genotype modified to contain multiple copies of a modified BAC in which EGFP
reporter gene is inserted immediately upstream of the coding sequence of the targeted
gene.
For further information and strain distribution please use the following URL:
http://www.mmrrc.org/strains/12003/012003.html
Characterization of Crym expression in the developing kidney
Figure 1.
Analysis of Crym-EGFP
expression in whole-embryos. Fluorescent
image detailing expression of EGFP in an
E16.5 embryo. This analysis detected wide
spread GFP expression in the developing
embryo including the skin, muscle, skeletal
elements and other regions of the embryo.
Figure 2. Expression of EGFP in the kidneys of
Crym-EGFP BAC transgenic mice. Top) Bright
field microscopy image detailing the expression of
Crym using whole-mount in situ hybridization in the
kidneys of E12.5 embryos (Image courtesy of
GUDMAP, Little Group). Middle) Fluorescent
microscopy image showing Crym-EGFP expression
in the kidney from E18.5 embryos. Note the
expression of GFP expression in the cap mesencyme.
Lower) Image represents a close-up detailing GFP
expression in the developing cap mesenchyme.
Characterization of Crym expression in the developing kidney
Figure 3. Confocal analysis of
Crym-EGFP expression in the
developing kidney. To further
delineate
and
localize
the
expression pattern of Crym-EGFP
in the kidney of E18.5 embryos, we
performed confocal analysis. This
image details the expression of
Crym-EGFP, which can be seen in
the cap mesenchyme. The tubules
of the kidney were labeled with Ecadherin. Crym (green) and Ecadherin (blue).
Confocal movie showing expression of Crym-EGFP in the developing kidney of E18.5
embryos. To further visualize Crym-EGFP expression, a file containing a movie that details
the expression of Crym-EGFP is provided. Strong Crym-EGFP expression can be detected in
the developing cap mesenchyme. The tubules of the kidney were labeled with E-cadherin
Crym (green) and E-cadherin (blue). The confocal images are available as a movie and can be
downloaded from http://www.gudmap.org/Resources/MouseStrains/index.html.
Methods
Tissue processing for confocal microscopy
Kidneys were dissected in phosphate buffered saline (PBS). The kidneys or the organ
explants were rocked for 1–2 h in 2% paraformaldehyde in PBS, washed twice with PBS,
and then rocked for 1–2 h in 100% methanol. The tissues were washed twice with cold
PBS containing 0.05% Tween-20 (PBT). Kidneys were bisected. Primary antibodies,
diluted to 1:250 to 1:400, were added to the tissues in 400 µL of PBT containing 2% goat
serum and incubated overnight with rocking. Tissues were washed with 5 exchanges of
PBT over 8 h with rocking. The secondary antibodies, diluted to 1:400 in PBT containing
2% goat serum, were added and incubated overnight. The tissues were again washed with
5 exchanges of PBT over 8 h. The tissue was washed for 5–10 min and mounted in a
depression slide in PBT before they were examined by confocal microscopy. The entire
procedure was performed at 4 °C with pre- cooled reagents.
The following primary antibodies were utilized: anti-Uvomorulin (E-cadherin, Sigma).
The secondary antibodies was Alexa 633-conjugated anti-rat secondary antibodies
(Molecular Probes).
Confocal imaging
The tissues were imaged with a Zeiss LSM510 equipped with an Argon (488 nm) and
two HeNe lasers (543 nm and 633 nm). We used a multi-track configuration, refractive
index correction, and automatic gain control. Approximately 2 µm thick optical sections
were obtained every 5 µm to a depth of at least 80 µm. The sections began at the surface
of the kidney and were on a plane tangential to it.
References
Gong S, Zheng C, Doughty ML, Losos K, Didkovsky N, Schambra UB, Nowak NJ,
Joyner A, Leblanc G, Hatten ME, Heintz N. A gene expression atlas of the central
nervous system based on bacterial artificial chromosomes Nature. 2003 Oct
30;425(6961):917-25.
GENSAT Project, Howard Hughes Medical Institute, The Rockefeller University, 1230
York Avenue, Box 260, New York 10021, USA."The Gene Expression Nervous System
Atlas (GENSAT) Project, NINDS Contracts N01NS02331 & HHSN271200723701C to
The Rockefeller University (New York, NY)."
Hartman HA, Lai, HL, Patterson LT. Cessation of renal morphogenesis in mice. Dev
Biology. 2007 310:379-387
Graw J. Genetics of crystallins: cataract and beyond. Exp Eye Res. 2009 Feb;88(2):17389.